DNA Replication, Repair, and Recombination

DNA Replication, Repair, and Recombination
Chapter 17 DNA Replication, Repair, and Recombination © 2016 Pearson Education, Inc.

DNA Replication, Repair, and Recombination
Cells must be able to accurately reproduce, or replicate, their genetic material at each cell division Without accurate replication, the genetic material of resulting cells would be riddled with errors Cells must also repair damage to their genetic material © 2016 Pearson Education, Inc. 2

17.1 DNA Replication All DNA in the nucleus of a parent cell must be duplicated and carefully distributed to the daughter cells The division process involves nuclear division (mitosis) and division of the cytoplasm (cytokinesis) Chromosomes that have duplicated consist of two sister chromatids © 2016 Pearson Education, Inc.

Separation of Sister Chromatids
The microtubules of the mitotic spindle separate the sister chromatids Each is now a full-fledged chromosome, and they move to opposite poles of the cell New nuclear envelopes form around the two sets of daughter chromosomes © 2016 Pearson Education, Inc.

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DNA Synthesis Occurs During S Phase
The events of mitosis (known as M phase) are only one phase of the cell cycle Cells spend most of their time between divisions, called interphase During interphase, the amount of nuclear DNA doubles during a specific time named S phase A time gap called G1 phase separates S phase from the previous M phase and a second gap, G2 phase, separates S phase from the next M phase © 2016 Pearson Education, Inc.

DNA Replication Is Semiconservative
The Watson and Crick model of DNA structure suggested a mechanism for how the base-paired structure could duplicate itself Their key suggestion was that one of the two strands of each new DNA molecule was derived from the parent molecule and the other strand was newly synthesized This is called semiconservative replication © 2016 Pearson Education, Inc.

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Two Other Models for DNA Replication
In the conservative model of DNA replication, the parent molecule is intact, and a second, completely new, copy is made In the dispersive model, each strand of the double helix is a mixture of old and newly synthesized segments © 2016 Pearson Education, Inc.

Demonstration of Semiconservative Replication
Matthew Meselson and Franklin Stahl (with Jerome Vinograd) showed that replication is semiconservative by using 14N and 15N to distinguish newly formed DNA strands from old Bacterial cells were grown in 15N medium for many generations to incorporate heavy nitrogen into their DNA, then transferred to 14N medium The strands were distinguished by equilibrium density centrifugation © 2016 Pearson Education, Inc.

Results of the Experiment
After one cycle of replication in the 14N medium, a band was observed intermediate between the heavy and light strands If the hybrid DNA was heated to separate the strands, one strand was “heavy,” and the other was “light” These results are consistent with semiconservative replication © 2016 Pearson Education, Inc.

Additional Experimental Results
After two cycles of replication, Meselson and Stahl observed two bands, one at the hybrid density and one entirely made of 14N-DNA Additional experiments used cells from bean roots and newly synthesized DNA labeled with 3H-thymidine These also support semiconservative replication © 2016 Pearson Education, Inc.

DNA Replication Is Usually Bidirectional
DNA replication is especially well understood in Escherichia coli Replication is very similar in prokaryotes and eukaryotes Early experiments to directly visualize DNA replication were carried out by John Cairns, using 3H-thymidine to label E. coli DNA © 2016 Pearson Education, Inc.

Replication Forks Cairns visualized the circular chromosomes by autoradiography; he observed replication forks These are formed where replication begins and then proceeds in bidirectional fashion away from the origin © 2016 Pearson Education, Inc.

Bacterial Replication
Replication forks move away from the origin, unwind the DNA, and copy both strands as they proceed This is called theta (Θ) replication and is observed in replication of circular DNA molecules The two copies of the replicating chromosome bind to the plasma membrane at their origins; when replication is complete, the cell divides by binary fission © 2016 Pearson Education, Inc.

Eukaryotic DNA Replication
In eukaryotes, replication of linear chromosomes is initiated at multiple sites, creating replication units called replicons The DNA of a typical chromosome may contain several thousand replicons, each 50,000 to 300,000 bp in length At each origin of replication, two replication forks synthesize DNA in opposite directions, forming a “replication bubble” © 2016 Pearson Education, Inc. 20

Replication Initiates at Specialized DNA Elements
The site where DNA replication initiates is known as an origin of replication, where synthesis is initiated by several groups of initiator proteins This sequence is AT rich and about 245 bp in length The sequence varies among bacterial species but contains recognizable, similar sequences, called consensus sequences © 2016 Pearson Education, Inc. 22

Replication Origins in S. cerevisiae
In budding yeast (Saccharomyces cerevisiae), the replication origin is called the autonomously replicating sequence (ARS) These elements are 100–150 bp long and contain a common 11 nucleotide core sequence, largely AT pairs Replication origins of multicellular eukaryotes are generally larger and more variable in sequence but also contain regions that are AT-rich © 2016 Pearson Education, Inc. 24

Replication Initiation in Bacteria
In E. coli, three enzymes, DnaA, DnaB, and DnaC, bind oriC and initiate replication DnaA binding to part of the oriC sequence results in unwinding of DNA To stabilize the single strands of DNA, SSB (single stranded binding protein) binds to the unwound regions DnaB is a DNA helicase, which unwinds the DNA strands as replication proceeds © 2016 Pearson Education, Inc. 25

Replication Initiation in Eukaryotes
Origins of replication recruit proteins that initiate the unwinding and replication of DNA First, a multisubunit protein complex called the origin recognition complex (ORC) binds the replication origin Next, the minichromosome maintenance (MCM) proteins bind the origin © 2016 Pearson Education, Inc. 27

Eukaryotic Replication
The MCM proteins include several DNA helicases that unwind the double helix; a set of proteins called helicase loaders recruit the MCM proteins At this point, all the DNA-bound proteins make up the pre-replication complex, and the DNA is “licensed” for replication © 2016 Pearson Education, Inc.

Replicons Are Not All Fired at the Same Time
Certain clusters of replicons replicate early during S phase, whereas others replicate later This is demonstrated by incubating cells during S phase with 5-bromodeoxyuridine (BrdU), which is incorporated into DNA in place of thymidine Active genes are replicated early during S phase, whereas inactive genes are replicated later © 2016 Pearson Education, Inc.

DNA Polymerases Catalyze the Elongation of DNA Chains
DNA polymerase is an enzyme that can copy DNA molecules Incoming nucleotides are added to the 3′ hydroxyl end of the growing DNA chain, so elongation occurs in the 5′ to 3′ direction Several other forms of DNA polymerase have been identified; the original is now called DNA polymerase I © 2016 Pearson Education, Inc.

DNA Polymerases Arthur Kornberg discovered the first DNA polymerase
Soon after, several other DNA polymerases were discovered © 2016 Pearson Education, Inc.

DNA Is Synthesized as Discontinuous Segments That Are Joined Together by DNA Ligase
DNA is synthesized in the 5′ to 3′ direction, but the two strands of the double helix are oriented in opposite directions One strand (the lagging strand) is synthesized in discontinuous fragments called Okazaki fragments The other (leading) strand is synthesized as a continuous chain © 2016 Pearson Education, Inc.

Okazaki’s Experiments
Reiji Okazaki isolated DNA from bacteria that were briefly exposed to a radioactive substrate incorporated into newly made DNA Much of the radioactivity was located in small fragments about 1000 nucleotides long With longer labeling, the radioactivity became associated with longer molecules; this conversion did not take place in bacteria lacking DNA ligase © 2016 Pearson Education, Inc.

Okazaki’s Observations Illustrate How Lagging Strand Synthesis Occurs
DNA synthesis from the lagging strand is synthesized in Okazaki fragments These are then joined by DNA ligase to form a continuous new 3′ to 5′ DNA strand Okazaki fragments are 1000–2000 nucleotides long in bacteria and viruses, but about one-tenth this length in eukaryotic cells © 2016 Pearson Education, Inc.

In Bacteria, Proofreading Is Performed by the 3′→ 5′ Exonuclease Activity of DNA Polymerase
About 1 of every 100,000 nucleotides incorporated during DNA replication is incorrect Such mistakes are usually fixed by a proofreading mechanism Almost all DNA polymerases have a 3′ → 5′ exonuclease activity © 2016 Pearson Education, Inc.

Proofreading Exonucleases degrade nucleic acids from the ends of the molecules Endonucleases make internal cuts in nucleic acid molecules The exonuclease activity of DNA polymerase allows it to remove incorrectly base-paired nucleotides and incorporate the correct base © 2016 Pearson Education, Inc.

RNA Primers Initiate DNA Replication
DNA polymerase can add nucleotides only to the 3′ end of an existing nucleotide chain Researchers implicated RNA in the initiation process based on several observations Okazaki fragments usually have short stretches of RNA at their 5′ ends © 2016 Pearson Education, Inc.

RNA Primers Initiate DNA Replication
Researchers implicated RNA in the initiation process based on several observations (cont’d) DNA polymerase can add nucleotides to RNA chains as well as DNA chains Cells contain an enzyme called primase that synthesizes short (~10 bases) chains of RNA using DNA as a template Primase is able to initiate RNA strands without a preexisting chain to add to © 2016 Pearson Education, Inc.

DNA Synthesis Requires RNA Primers
The observations led to the conclusion that DNA synthesis is initiated by the formation of short RNA primers These are synthesized by primase using a single DNA strand as the template In E. coli, primase is inactive unless accompanied by six other proteins, forming a complex called a primosome © 2016 Pearson Education, Inc.

The Process of DNA Synthesis
Once the RNA primer is made, DNA polymerase III (or DNA polymerase α, followed by δ or ε, in eukaryotes) adds deoxynucleotides to the 3′ end of the primer For the leading strand, just one primer is needed, but the lagging strand needs a series of primers to initiate each Okazaki fragment When the DNA chain reaches the next Okazaki fragment, the RNA is degraded and replaced with DNA; adjacent fragments are joined together by DNA ligase © 2016 Pearson Education, Inc.

The DNA Double Helix Must Be Locally Unwound During Replication
During DNA replication, the two strands of the double helix must unwind at each replication fork Three classes of proteins facilitate the unwinding: DNA helicases, topoisomerases, and single- stranded DNA binding proteins DNA helicases are responsible for unwinding the DNA, using energy from ATP hydrolysis © 2016 Pearson Education, Inc.

Helicases The DNA double helix is unwound ahead of the replication fork, the helicases breaking the hydrogen bonds as they go In E. coli, at least two different helicases are involved; one attaches to the lagging strand and moves 5′ → 3′, whereas the other attaches to the leading strand and moves 3′ → 5′ Both are part of the primosome © 2016 Pearson Education, Inc.

Single-Stranded DNA Binding Protein
Once strand separation has begun, molecules of SSB (single-stranded DNA binding protein) move in quickly and attach to the exposed single strands They keep the DNA unwound and accessible to the replication machinery When a segment of DNA has been replicated, the SSB molecules fall off and are recycled © 2016 Pearson Education, Inc.

Topoisomerases The unwinding of the helix would create too much supercoiling if not for topoisomerases These enzymes create swivel points in the DNA molecule by making and then quickly sealing double-stranded or single-stranded breaks Of the ~10 topoisomerases in E. coli, the key enzyme for DNA replication is gyrase © 2016 Pearson Education, Inc.

DNA Unwinding and DNA Synthesis Are Coordinated on Both Strands Via the Replisome
Starting at the origin of replication, the machinery at the replication fork adds proteins required for synthesizing DNA These are DNA helicase, DNA gyrase, SSB, primase, DNA polymerase, and DNA ligase The proteins involved in replication are closely associated in a large complex called a replisome © 2016 Pearson Education, Inc.

The Replisome The replisome is about the size of a ribosome
The activity and movement of the replisome is powered by nucleoside triphosphate hydrolysis As the replisome moves along the DNA, it must accommodate the fact that DNA is being produced on both leading and lagging strands © 2016 Pearson Education, Inc.

The Trombone Model A key element of the replisome is the folding of the lagging strand template into a loop This model for how the replisome works is called the “trombone” model A sliding clamp protein that attaches to a DNA polymerase catalytic subunit allows the polymerase to “process” along the DNA without falling off © 2016 Pearson Education, Inc.

Leading and Lagging Strands
The leading and lagging strands differ regarding how long the sliding clamp and associated polymerase remain attached It can remain associated with the leading strand throughout replication On the lagging strand, as each Okazaki fragment is completed, the polymerase detaches, and the sliding clamp must be reloaded © 2016 Pearson Education, Inc.

Eukaryotes Disassemble and Reassemble Nucleosomes as Replication Proceeds
Eukaryotes have much of the same replication machinery found in prokaryotes For example, a DNA clamp protein acts along with DNA polymerase; one of these is called proliferating nuclear cell antigen (PCNA) PCNA is a clamp protein for DNA polymerase δ © 2016 Pearson Education, Inc.

Replication Factories and Chromatin Remodeling
Studies addressing how many origins of replication can be coordinated suggest that immobile structures called replication factories synthesize DNA as chromatin fibers are fed through them Unfolding chromatin fibers ahead of the replication fork is facilitated by chromatin remodeling proteins that loosen nucleosome packing © 2016 Pearson Education, Inc.

Chromatin Remodeling Proteins
After a stretch of DNA is replicated, nucleosomes are reassembled on the newly formed strands Nucleosome assembly protein-1 (Nap-1) and chromatin assembly factor-1 (CAF-1) are examples of chromatin assembly proteins The dynamic disassembly/reassembly allows nucleosome association with DNA throughout the replication process © 2016 Pearson Education, Inc.

Telomeres Solve the DNA End-Replication Problem
Linear DNA molecules have a problem in completing DNA replication on the lagging strand because primers are required Each round of replication would end with the loss of some nucleotides from the ends of each linear molecule Eukaryotes solve this problems with telomeres, highly repeated sequences at the ends of chromosomes © 2016 Pearson Education, Inc.

Telomeres and Telomerase
Human telomeres have 100 to 1500 copies of TTAGGG at the ends of chromosomes These noncoding sequences ensure that the cell will not lose important genetic information if DNA molecules shorten during replication A polymerase called telomerase can catalyze the addition of repeats to chromosome ends © 2016 Pearson Education, Inc.

Telomerase Function Telomerase is composed of protein and RNA
In the protozoan Tetrahymena, the RNA component of the telomerase (3′—AACCCC—5′) is complementary to the telomere repeat sequence (5′—TTGGGG—3′) This enzyme-bound RNA acts as a template for adding the DNA repeat sequence to the telomere ends © 2016 Pearson Education, Inc.

Protecting Chromosome Ends
After telomeres are lengthened by telomerase, telomere capping proteins bind to the exposed 3′ end to protect from degradation In many eukaryotes, the 3′ ends of the DNA also loop back and base-pair with the opposite strand to form a protective closed loop In multicellular organisms, telomerase function is restricted to germ cells and a few other types of actively proliferating cells © 2016 Pearson Education, Inc.

Most Cells Have a Limited Life Span
Telomere shortening occurs with each cell division in most cells As a result, telomere length is a counting device for how many times a cell has divided; if a cell divides too many times, telomeres could be lost Cells at risk of loss of telomeres undergo apoptosis, programmed cell death © 2016 Pearson Education, Inc.

Telomeres, Aging, and Disease
Immortalized cell lines, such as HeLa cells, produce telomerase and can be passaged indefinitely Cell death triggered by a lifetime of telomere shortening is thought to contribute to some of the degenerative diseases associated with human aging Scientists speculate that telomerase-based therapy may one day be used to combat symptoms of human aging © 2016 Pearson Education, Inc.

Telomeres, Aging, and Disease (continued)
Telomerase has been detected in almost all types of human cancers Proteins that bind the tandem repeat DNA in telomeres recruit telomere capping proteins to protect the single-stranded DNA at the ends from damage Patients with Werner syndrome lack a telomere cap protein (WRN) and exhibit premature signs of aging © 2016 Pearson Education, Inc.

17.2 DNA Damage and Repair DNA must be accurately passed on to daughter cells In addition to ensuring that replication is faithful, this also means that DNA alterations must be repaired DNA alterations, or mutations, can arise spontaneously or through exposure to environmental agents © 2016 Pearson Education, Inc.

Mutations Can Occur Spontaneously During Replication
During DNA replication, some types of mutations occur through Spontaneous mispairing of bases due to transient formation of tautomers Slippage during replication Spontaneous damage to individual bases © 2016 Pearson Education, Inc.

DNA Tautomers Mispairing of DNA nucleotides due to presence of tautomers is the most common form of spontaneous replication error Tautomers are rare, alternate resonance structures of nitrogenous bases In this form, a base can pair in a nonstandard way in a process known as a tautomeric shift The result is a new daughter strand that carries an incorrect base at that position © 2016 Pearson Education, Inc.

Trinucleotide Repeats
Spontaneous replication errors can occur in regions with repetitive DNA One example involves trinucleotide repeats, which are susceptible to strand slippage In this process, DNA polymerase replicates a short stretch of DNA twice Several well-characterized human diseases, trinucleotide repeat disorders, involve accumulation of various trinucleotide repeats © 2016 Pearson Education, Inc.

Depurination and Deamination
Another reaction that can occur spontaneously involves chemical modification of bases Depurination, the loss of a purine base, and deamination, the removal of a base’s amino group, are the most common A human cell may undergo thousands of depurinations each day, and about 100 deaminations Failure to repair these can lead to base changes in the DNA sequence © 2016 Pearson Education, Inc.

Mutagens Can Induce Mutations
DNA damage can be caused by mutation-causing agents, mutagens Environmental mutagens fall into two categories: chemicals and radiation Mutation can also be induced by mobile genetic elements, such as found in viruses, or transposable elements (transposons) Mutagenic chemicals alter DNA structure through a variety of mechanisms © 2016 Pearson Education, Inc.

DNA Damage by Chemical Mutagens
Base analogues resemble nitrogenous bases and are incorporated into DNA Base-modifying agents react chemically with DNA bases to alter their structures, forming DNA adducts Intercalating agents insert themselves between adjacent bases, distorting DNA structure © 2016 Pearson Education, Inc.

Base Analogues Base analogues, structurally similar to one of the DNA nucleotides, can be incorporated into a DNA molecule during replication An example is 5-bromodeoxyuridine (BrdU) with pairing properties similar to thymine Wherever it is incorporated into DNA, when the DNA is replicated, an A is incorporated into the new strand © 2016 Pearson Education, Inc.

Base-Modifying Agents
Several mutagens act by chemically modifying a base that will then mispair at the next replication Ethyl methansulfonate (EMS) adds ethyl groups to bases, while nitrosoguanidine adds methyl groups Nitrous acid (HNO2) dramatically increases the likelihood of deamination Other agents add bulky DNA adducts to DNA Aflatoxin B1 attaches to guanine, leading to depurination © 2016 Pearson Education, Inc.

Intercalating Agents Intercalating agents (such as proflavin, acridine orange, benzo(a)pyrene) insert into the DNA double helix They alter the shape of the double helix, leading to small nicks in the DNA When repaired, there may be additions or deletions of nucleotides Ethidium bromide is a common fluorescent dye (and intercalating agent) used for gel electrophoresis of DNA © 2016 Pearson Education, Inc.

Radiation Ultraviolet radiation alters DNA by triggering pyrimidine dimer formation—covalent bonds between adjacent pyrimidine bases X-rays and related types of radiation, called ionizing radiation, remove electrons from molecules and generate highly reactive intermediates that damage DNA © 2016 Pearson Education, Inc.

DNA Repair Systems Correct Many Kinds of DNA Damage
A variety of mechanisms have evolved for DNA repair The strategies depend on how severe the damage is and whether or not the cell is undergoing division © 2016 Pearson Education, Inc.

Light-Dependent Repair
Pyrimidine dimers can be directly repaired in a light-dependent process called photoactive repair It depends on the enzyme photolyase, which catalyzes breakage of bonds between thymine dimers The energy for this repair is provided by visible light © 2016 Pearson Education, Inc.

Base Excision Repair Excision repair pathways are classified into two types: base excision repair and nucleotide excision repair Base excision repair corrects single damaged bases For example, deaminated bases are detected by DNA glycosylases, which recognize and remove the base by cleaving the bond between the base and the sugar © 2016 Pearson Education, Inc.

Base Excision Repair (continued)
The sugar with the missing base is then recognized by a repair endonuclease (AP endonuclease) that detects depurination It breaks the phosphodiester backbone to one side of the sugar, and a second enzyme removes the sugar DNA polymerase then synthesizes the correct new base, and DNA ligase seals the nick in the DNA © 2016 Pearson Education, Inc.

Nucleotide Excision Repair
Nucleotide excision repair (NER) uses proteins that detect distortions in the DNA helix and recruit NER endonuclease (or excinuclease) that cuts the DNA backbone on either side of the lesion Helicase unwinds the DNA between the nicks and frees it from the DNA; DNA polymerase and ligase complete the repair © 2016 Pearson Education, Inc.

The NER System Is Versatile
The nucleotide excision repair system detects and corrects many types of DNA damage Sometimes it is recruited to regions where transcription is stalled because of DNA damage; this is called transcription-coupled repair People with xeroderma pigmentosum must stay out of the sun because of mutations that prevent them from carrying out NER © 2016 Pearson Education, Inc.

Mismatch Repair Errors remaining after DNA replication are repaired by excision repair, in which abnormal nucleotides are removed and replaced E. coli has nearly 100 genes that code for proteins involved in this process Excision repair works by a basic three-step process © 2016 Pearson Education, Inc.

Methylation in Mismatch Repair
DNA methylation does not occur immediately after DNA replication Therefore, mismatch repair systems can distinguish the original DNA (methylated) from the newly made strand (unmethylated) The incorrect nucleotide in the newly made strand is excised and replaced © 2016 Pearson Education, Inc.

Process of Mismatch Repair
A protein known as MutS detects the mismatch A repair endonuclease called MutH introduces a nick in the unmethylated strand An exonuclease removes the incorrect nucleotides from the nicked strand, and these are replaced with the correct sequence © 2016 Pearson Education, Inc.

Error-Prone Repair: Translesion Synthesis
Sometimes, DNA damage is too severe for the preceding types of repair Bacteria and eukaryotes have evolved mechanisms that serve as a last-ditch effort to repair DNA In E. coli, this process involves the SOS system, activated when a replication fork stalls due to damage of the DNA © 2016 Pearson Education, Inc.

The SOS System in E. coli The DNA in front of the stalled DNA polymerase continues to be unwound A protein called RecA joins SSB on this single strand RecA leads to expression of special DNA polymerases called bypass polymerases These continue the DNA replication despite the damage Once the damage is passed, DNA polymerase III can resume the replication © 2016 Pearson Education, Inc.

Translesion Synthesis
In eukaryotes, specialized bypass polymerases carry out translesion synthesis Though it is sometimes prone to error, this type of synthesis can sometimes produce new strands from which the damage has been eliminated For example, polymerase η can catalyze synthesis across a thymine dimer, correctly inserting two new adenines in the new strand © 2016 Pearson Education, Inc.

Double-Strand Break Repair
Double-strand breaks cleave DNA into two fragments It is difficult for the repair system to identify and rejoin the correct broken ends without loss of nucleotides Two pathways are used: nonhomologous end- joining and homologous recombination © 2016 Pearson Education, Inc.

Nonhomologous End-Joining
Nonhomologous end-joining uses a set of proteins that bind to ends of broken DNA fragments and join them together This is error-prone because nucleotides can be lost from the broken ends, and there is no way to ensure the correct DNA fragments are joined © 2016 Pearson Education, Inc.

Process of Nonhomologous End-Joining
A double-stranded break in the DNA is detected by Ku70 and Ku80 proteins, which bind one another and fit over the broken DNA ends The ends of the break are trimmed by nucleases The break is sealed by DNA ligase IV This process is used only when the existing DNA sequence cannot be used as a template for repair © 2016 Pearson Education, Inc.

Synthesis-Dependent Strand Annealing
Synthesis-dependent strand annealing (SDSA) depends on the fact that once DNA synthesis is complete, each chromosome has two sister chromatids Thus if one chromatid incurs a double-strand break, there is a second intact copy of the same DNA available to guide repair © 2016 Pearson Education, Inc.

Process of Synthesis-Dependent Strand Annealing
SDSA begins with trimming of the break by nucleases and recruitment of the Rad51 protein to strands at the break Strands search for homologous sequences on the sister chromatid during a process called strand invasion The invading strand displaced the DNA of the sister chromatid, creating a D loop © 2016 Pearson Education, Inc.

Process of Synthesis-Dependent Strand Annealing (continued)
The D loop structure allows replication of the single strand from the broken DNA Bits of repaired DNA then dissociate from the D loop and are ligated to the broken chromatid, leading to its repair © 2016 Pearson Education, Inc.

Homologous Recombination
Homologous recombination involves the process of crossing over, genetic exchange between DNA molecules with extensive sequence similarity If the DNA molecule from one chromosome is broken, the homologue is available as a template to guide accurate repair © 2016 Pearson Education, Inc.

Process of Homologous Recombination
The break in the DNA is detected and the ends trimmed As with SDSA, strand invasion occurs, but involving both strands DNA synthesis fills in DNA on both strands using the intact pieces of DNA as a template The result is a Holliday junction, a crossed structure, which is resolved to generate two separate stands of repaired DNA © 2016 Pearson Education, Inc.

Result of Homologous Recombination
One possible result is permanent exchange of DNA between the broken DNA of one homologue and the intact DNA of the other The other possibility is repair of the DNA without exchange In this case, the repaired chromosome has the same sequence as the undamaged one This process is known as gene conversion © 2016 Pearson Education, Inc.

17.3 Homologous Recombination and Mobile Genetic Elements
A widespread exchange of chromatin occurs during the process of meiosis © 2016 Pearson Education, Inc. 114

Homologous Recombination Is Initiated by Double-Stranded Breaks in DNA
Homologous recombination is more complicated that a simple breakage-and-exchange model This model has been superseded by a model involving double-stranded breaks in the DNA © 2016 Pearson Education, Inc. 115

Double-Stranded Breaks and the Formation of Crossovers
In the current model, the initial step of recombination is the asymmetric cleavage of one chromatid by the dimeric protein Spo11 Spo11 recruits two other proteins that trim the single strands Rad51 stabilizes the single stranded DNA, as it does in DNA repair © 2016 Pearson Education, Inc. 116

Steps of Recombination
The stabilized DNA strands invade a complementary region on another homologous piece of DNA, forming a D loop The invading strand pairs with the DNA in the D loop The Holliday junction forms © 2016 Pearson Education, Inc.

Strand Extension Once the Holliday junction is formed, DNA synthesis leads to strand extension, or branch migration The D loop is displaced and can pair with more single-stranded DNA, which involves other proteins, such as Rad52 and Rad59 Gaps in the single stranded DNA are repaired via DNA polymerase and ligase © 2016 Pearson Education, Inc. 119

Double Holliday Junction
Finally, the 3′ end of the invading strand connects with the 5′ end of one of the original single-stranded segments This results in a double Holliday junction © 2016 Pearson Education, Inc. 120

Holliday Junction Resolution
Holliday junctions are transitory structures that must be resolved (and the homologous chromosomes disconnected) before cell division The junction can be cleaved, or resolved, in either of two ways © 2016 Pearson Education, Inc. 121

Holliday Junction Resolution (continued)
If the junction is resolved via same-sense resolution, crossing over does not occur, though DNA molecules have a noncomplementary region near the site of the junction Opposite-sense resolution results in crossing over The latter is more common © 2016 Pearson Education, Inc. 122

Transposons Are Mobile Genetic Elements
Recombination is one way organisms maintain genetic diversity Another type of genetic exchange involves mobile genetic elements, called transposable elements or transposons The first evidence for such movable genetic elements came from pioneering work by the maize (corn) geneticist Barbara McClintock in the 1940s and 1950s © 2016 Pearson Education, Inc.

Movement of Transposons
Transposon movement does not require sequence similarity with the insertion site Transposons encode all or many of the components they need to move within a genome DNA-only transposons act only through DNA and the proteins they encode Retrotransposons act through an RNA intermediate step © 2016 Pearson Education, Inc. 125

Transposons Differ Based on Their Autonomy and Mechanism of Movement
Autonomous transposable elements encode their own transposase (enzyme required for movement) Nonautonomous transposable elements lack a transposase genes and need the help of other autonomous elements to move This process is sometimes referred to as “mobilizing” the element © 2016 Pearson Education, Inc.

Two Mechanisms of Transposition
In replicative transposition, the transposon is copied from the current site and the new copy inserted at the new site (a “copy-and-paste” mechanism) In conservative transposition, the transposon is excised from the original site and moves to a new site in the genome (a “cut-and-paste” mechanism) © 2016 Pearson Education, Inc. 127

Bacterial Transposons Can Be Composite or Non-composite
Composite transposons carry IS (insertion- sequence) elements Different IS elements vary in length and structure, but all encode transposases and begin and end with short inverted repeats Noncomposite transposons do not rely on IS elements Instead, they have short, inverted repeat sequences at their ends and a transposase gene in the middle © 2016 Pearson Education, Inc.

Mechanism of Insertion at a New Location
When a transposase gene is used to produce transposase enzyme, the transposon can move to a new site © 2016 Pearson Education, Inc.

Steps of Transposon Movement
Transposase makes a staggered cut in the DNA at the new insertion site The transposon inserts between the staggered ends The DNA repair machinery of the bacterium seals the remaining single-stranded gaps The result is two duplicate sequences on either side of the inserted transposition, called target site duplication © 2016 Pearson Education, Inc. 132

Eukaryotes Also Have Transposons
Other eukaryotes (besides maize) have DNA-only transposons One of the best studies is the P element of the fruit fly, Drosophila When P elements insert into genes at random, they can disrupt the function of the gene, a process called transposon tagging © 2016 Pearson Education, Inc.

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